Figure 2: Western Blot of His-tagged C. tetani antigen with anti His-tag HRP Conjugate. The soluble fraction of the overexpressed protein was used for this Western Blot. The expected molecular weight is 50 kDa. Anti- His Tag HRP conjugated antibody was used in a 1:1000 dilution in blocking buffer.

The antigen used for detection of anti-tetanus antibodies is derived from the tetanus neurotoxin and commonly used for in vitro testing. For preparation of the DiaCHIP, this antigen had to be overexpressed in E. coli and purified from whole cell lysate. To verify the successful purification of the antigen, Western Blot analysis was performed (figure 2). The usage of an antibody against the N-terminal His-tag revealed a protein with a molecular weight of about 50 kDa. This correlates with the expected molecular weight of the antigen ¹⁾.

Figure 3: ELISA for the tetanus antigen with blood samples taken after the immunization. The graph shows the absorption over time for TMB. The red line shows the binding of anti-tetanus antibodies out of the blood serum to purified tetanus antigen and the blue line shows the binding of HRP-Streptavidin to bBSA. The grey lines show the corresponding negative controls.

We performed an ELISA to investigate, whether the blood samples we obtained contain antibodies that bind specific to our purified tetanus antigen. Therefore, we spotted tetanus antigen and bBSA of the same concentration (12.5 µg/mL) and 2 times PBS as negative controls. The tetanus antigen and one PBS well were incubated with 5 mg/mL BSA, blood serum, and HRP-labeled anti-human for 1 h, respectively. The bBSA and the other PBS well were incubated with HRP-labeled Streptavidin (0.5 µg/mL) for 1  h (positive control). After each step the wells were washed with PBS. To visualize the binding, Tetramethylbenzidine was added and the absorption at 650 nm was measured in a plate reader over time (figure 3). The absorption signal for tetanus is even higher than the positive control, which illustrates the successful binding of antibodies from the blood to tetanus antigen.

Figure 4: Binding curves for the detection of the binding of anti-tetanus antibodies from blood serum via iRIf shown in figure 1.

After the validation of the antigen antibody binding for tetanus by ELISA we wanted to see if we can detect this interaction also with iRIf. Therefore we first expressed our Clostridium tetani antigen and GFP in E.coli. The purified His-tagged proteins then were spotted on a PDITC surface. The slide with the immobilized proteins was flushed with human blood serum, and anti-human antibody. Strong binding was detected for the tetanus antigen spots, while the GFP spots only exhibited minimal binding (see figure 1A-D). This indicates that components contained in the blood serum bind specifically to the tetanus antigen. The subsequent binding of anti-human antibody to the tetanus antigen spots (figure 4) confirms the presents and binding of human antibodies to the spotted tetanus antigens. These results successfully demonstrate that it is possible to detect the binding of antibodies from blood serum to antigens immobilized, on a surface.

Figure 5: Quotient picture of iRIf measurement. Line A was flushed with serum taken before vaccination against tetanus. Line B was flushed with serum taken 2 weeks after vaccination. Spots from left to right show: GFP, neg. control, Tetanus antigen.

With an additional experiment we wanted to see if we can differentiate the vaccination status of patients with our system. Therefore we used blood serum samples, taken before and two weeks after immunization against tetanus. A slide with immobilized tetanus antigen and GFP was prepared (figure 5). It was consecutively flushed with both blood sera. The blood serum taken after vaccination exhibits a much more intense binding to the tetanus spot than the serum taken before binding. Unfortunately we did not had enough anti-human antibody to proof further the presents of bound human antibodies from blood serum.

Figure 6: Quotient pictures of slides flushed with blood serum taken before and after vaccination. (A) Schematic pattern of proteins immobilized on a glass slide. (B) Quotient picture of iRIf measurement flushed with anti-GFP and blood serum taken before vaccination . (C) Quotient picture of iRIf measurement in which the slide was flushed with anti-GFP and blood serum five weeks after vaccination.

While the outcome of this first experiment looked promising for our future application to determine the vaccination status of patients, further experimentation did not confirm these initial results. We were still able to detect binding of both blood sera to the tetanus antigen, but no difference in binding intensity was visible (See figure 6B and C). To test the assumption that the used blood was not fresh enough and therefore lost some of its binding capabilities a fresh blood sample five weeks after vaccination was taken for the experiment. To determine whether the identification of the vaccination status is possible with our system further experimentation needs to be done.

We could successfully show that the cell-free expression of His-GFP results in functional GFP proteins that are bound by anti-GFP antibody during an iRIf measurement. The DNA coding for GFP fused to a 10x His-tag was added to the DiaMIX and expression was performed for 2 h. The DiaMIX containing cell-free expressed His-GFP was then spotted on an iRIf slide with a Ni-NTA surface by hand (figure 1). The spots were incubated on the slide overnight.

Figure 1: Specific Ni-NTA surface on iRIf glass slide.

This specific surface allows binding of the expressed His-GFP via the His-tag while all the other proteins present in the DiaMIX are not bound to the surface. This step was followed by the blocking and washing protocol to prevent further unspecific binding. The iRIf slide was then flushed with an anti-GFP antibody solution, to analyze the binding to the cell-free expressed His-GFP. As a positive control we spotted a purified His-GFP onto the surface as well as DiaMIX that did not contain any DNA as negative control figure 2).
Find out more about the preparation of the DiaCHIP by producing a protein microarray from a DNA template using cell-free expression.

The measurement shows a high signal for the cell-free expressed His-GFP and the positive control as can also be seen in the respective binding curve shown in figure 3.

Figure 2: Measurement of cell-free expressed GFP on Ni- NTA surface. The presence of GFP is detected with GFP antibodies within an iRIf measurement The spot on the right is the cell-free expressed His-GFP protein spotted onto the Ni-NTA surface. The spot on the left shows the positive control, His-GFP expressed in E. coli, purified and spotted onto the Ni-NTA surface. The negative control represents the DiaMIX without DNA. A clear iRIf signal is detected for the positive control and the cellfree expressed GFP.

Figure 3: Binding curve of anti-GFP binding to cell-free expressed His-GFP on a Ni-NTA surface. Cell-free expressed GFP-His was spotted onto a specific Ni-NTA surface and flushed with anti-GFP. The binding curve indicated a binding event at the respective spot.

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To test our device we aimed at reproducing measurements that we were previously able to perform in our commercial device. For this reason we focused on using two antibodies: anti-GFP and polyclonal anti-rabbit. GFP and rabbit derived anti-HCV (Hepatitis C Virus) antibody were used as antigens in this experiment. Note that the anti-HCV antibody only served as binding partners for the anti-rabbit antibodies, since no HCV proteins were used in this experiment. The antigens were spotted onto an iRIf slide with an APTES/PDITC surface. The spots on the slide were produced by pipetting 3.5 µl [1 mg/ml] rabbit-anti-HCV protein and 3.5 µl [1 mg/ml] GFP onto the slide and incubated overnight. After incubation the slide was blocked for 30 min in BSA solution. A Canon 50D camera was used to record the measurement and was set to take one picture every 5 seconds. The exposure time was set in order for the pixels in the image to be approx. 80% of maximum light saturation before the solution was flushed onto the chip. The antibody solutions were pipetted into the flow-chamber without the use of any microfluidic device. Instead, a syringe was loaded with 660 µl [5 µg/ml] anti-GFP antibody solution (diluted in PBS) and connected to the input pipe of our device. The content of the syringe was then slowly released via this pipe into the chamber of the device by gently dispensing the solution from the syringe. When the whole volume was run over the chip, the process was repeated with 660 µl [5 µg/ml] anti-rabbit antibody solution in the same way. The injection of both solutions was performed during approximately 45 minutes.

Figure 3: Quotient picture of the same measurement with favorable light conditions.

The quotient pictures show all intensity shifts that occurred during the measurement. Two left spots: GFP spots at which an intensity shift occurred during anti-GFP flush. Right spot: anti-HCV antibody (derived from rabbit), at which an intensity shift occurred during the flush with anti-Rabbit.

Figure 4: Comparison of two quotient pictures of our DiaCHIP with the professional setup. Left: Professional device; Right: Our device. Both quotient pictures show the binding events that occurred during an iRIf measurement with anti-GFP at the GFP spots.

Video 1 shows the results of the measurement. Both, binding of anti- GFP and anti-rabbit to the corresponding antigen spots, could be observed. Due to the fact that the experiment was performed on our DiaCHIP device which was built with a transparent casing, fluctuations in surrounding light had a strong, detrimental influence on the measurement quality. To minimize the influence of the unstable surrounding light, the resulting pictures had to be averaged over 10 pictures each. This in turn led to a more stable light situation, however the signal strength dropped as a consequence. Figure 3 shows a quotient picture of the measurement where the light situation was temporarily appropriate. The problem of surrounding light scattering into the device can of course be overcome using a non-transparent casing. An evaluation of how well our device performs in comparison with the professional device was not performed, mainly due to time constraints. However our results hint that we get comparable results for strong binding such as GFP/anti-GFP, as can be seen in figure 4.

Figure 2: Cell-free expressed His-GFP spotted on a PDITC slide. Cell-free expression was performed for 2 hours at 37°C. The positive control was purified His-GFP, the negative control was a sample of cell-free expression mix not containing DNA.

For cell-free protein expression lots of different proteins, like RNA-polymerases, ribosomes and other E. coli proteins are essential. During the process of cell-free expression all these proteins are, besides the target protein, also present in the microfluidic chamber. To have a sufficient amount of target protein immobilized on the chip we established a specific surface chemistry on the glass slide. After testing several tag systems we established a Ni-NTA surface. Using a Ni-NTA covered glass slide (figure 1) we could increase the amount of bound cell-free expressed His-GFP compared to an unspecific surface (figure 2). In both experiments 1.5 µg of self purified GFP-His was used as positive control. The cell-free expression mix without DNA is supposed to result in no target protein and served as negative control. The cell-free reactions were performed for 2 hours at 37°C in a seperate tube prior to pipetting the samples by hand onto the surfaces and incubated on the slide overnight. Before measurment in the iRIf device, the optical detection method we use, the slides were blocked with BSA and then flushed with anti-GFP antibodies. An increase in light intensity where the spotted protein was located represents a binding event. Due to less unspecific binding of proteins to the surface, the interaction of anti-GFP antibodies with cell-free expressed His-GFP is higher on the Ni-NTA surface.

Our DiaMIX is a complex system consisting of several individually adjusted components that allow us to make use of the cells own production system, without the tedious drawbacks of normal cell-based protein expression. As a main component of the DiaMIX the E. coli lysate contains the necessary transcription and translation apperatus. The lysate is complemented by additives needed for energy regeneration, buffering, and generation of the protein. An overview of cell-free expression systems and its components can be found on our project pages.
As ultimate evaluation, we compared the DiaMIX to a commercial kit as seen in figure 1.

Both expression systems successfully expressed the applied vector.
Furthermore, the cell-free expression of GFP with our cell-free mix could be verified in an iRIf measurement.
To obtain these final results, a lot of optimization and testing had to be done. Want to know more about how we established the DiaMIX? You can find detailed information about the most important experiments on the cell-free expression results page.